腺相关病毒载体pAGX(+)的构建

Construction of an adeno-associated virus vector pAGX(+)

目的 构建腺相关病毒 (AAV)载体并进行鉴定 ,以介导真核细胞的基因传递和表达。方法 以基因重组技术构建AAV载体 ,用Southern杂交、狭缝杂交以及激光共聚焦显微镜等鉴定构建的AAV载体。结果 成功地获得了重组AAV表达载体pAGX( + ) ,藉报告基因EYFP鉴定pAGX( + ) ,见pAEYFP经包装的重组AAV储存液的感染滴度达 1 .3 9× 1 0 7TU(transmissionunit) /ml、复制滴度达1 .94× 1 0 11颗粒 /ml。Southern杂交表明EYFP基因获得成功拯救。rAAV/EYFP颗粒成功转导COS 7细胞 ,EYFP获得表达 ,并整合入COS 7细胞基因组 ,而藉质粒载体pEYFPCMV介导的转移 ,EYFP未能整合。结论 成功地构建了一个AAV载体 ,并成功地介导了基因的转移、表达和整合。

Objective To construct an adeno associated virus vector for gene delivery to and gene expression in mammalian cells. Methods Gene recombinant technique for the construction of the vector, Southern blot, slot blot and laser confocal microscopy for determining the efficiency of EYFP gene rescue, gene transfer,expression and integration into host genome mediated by pAGX(+). Results An adeno associated virus vector named pAGX(+) was successfully constructed. The infection titer and the replication titer of rAAV/EYFP virion was 1.39×10 7 TU/ml and 1.94×10 11 particles/ml, respectively. Southern blot proved that the EYFP gene was successfully rescued. EYFP gene was successfully transducted to and expressed in mammalian cell line COS 7 cells. Southern analysis showed that the EYFP gene was integrated into the genomic DNA of COS 7 cells mediated by the transduction of rAAV/EYFP. On the contrary, the plasmid pEYFP CMV mediated EYFP gene transfer did not show EYFP gene integration. Conclusion An adeno associated virus vector pAGX(+) was successfully constructed, and the transfer, expression and integration of a gene were successfully mediated in mammalian cells by the rAAV.